Exchange-dependent relaxation in the rotating frame for slow and intermediate exchange - modeling off-resonant spin-lock and chemical exchange saturation transfer

Chemical exchange observed by NMR saturation transfer (CEST) and spin-lock (SL) experiments provide an MRI contrast by indirect detection of exchanging protons. The determination of the relative concentrations and exchange rates is commonly achieved by numerical integration of the Bloch-McConnell equations. We derive an analytical solution of the Bloch-McConnell equations that describes the magnetization of coupled spin populations under radiofrequency irradiation.As CEST and off-resonant SL are equivalent, their steady-state magnetization and dynamics can be predicted by the same single eigenvalue: the longitudinal relaxation rate in the rotating frame R1rho. For the case of slowly exchanging systems, e.g. amide protons, the saturation of the small proton pool is affected by transverse relaxation (R2b). It turns out, that R2b is also significant for intermediate exchange, such as amine- or hydroxyl-exchange or paramagnetic CEST agents, if pools are only partially saturated. We propose a solution for R1rho that includes R2 of the exchanging pool by extending existing approaches, and verify it by numerical simulations. With the appropriate projection factors, we obtain an analytical solution for CEST and SL for nonzero R2 of the exchanging pool, whilst considering the dilution by direct water saturation across the entire Z-spectra. This allows the optimization of irradiation parameters and the quantification of pH-dependent exchange rates and metabolite concentrations. In addition, we propose evaluation methods that correct for concomitant direct saturation effects. It is shown that existing theoretical treatments for CEST are special cases of this approach

Systematic study of constitutive cyclo-oxygenase-2 expression: role of NFκB and NFAT transcriptional pathways

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system

The influence of an infection with Ascaris suum on the intestinal microbiota of the host

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Possible mechanisms of host resistance to Haemonchus contortus infection in sheep breeds native to the Canary Islands

Haemonchus contortus appears to be the most economically important helminth parasite for small ruminant production in many regions of the world. The two sheep breeds native to the Canary Islands display distinctly different resistant phenotypes under both natural and experimental infections. Canaria Hair Breed (CHB) tends to have significantly lower worm burden and delayed and reduced egg production than the susceptible Canaria Sheep (CS). To understand molecular mechanisms underlying host resistance, we compared the abomasal mucosal transcriptome of the two breeds in response to Haemonchus infection using RNAseq technology. The transcript abundance of 711 and 50 genes were significantly impacted by infection in CHB and CS, respectively (false discovery rate <0.05) while 27 of these genes were significantly affected in both breeds. Likewise, 477 and 16 Gene Ontology (GO) terms were significantly enriched in CHB and CS, respectively (P < 1.0 × 10(−4)). A broad range of mechanisms have evolved in resistant CHB to provide protection against the parasite. Our findings suggest that readily inducible acute inflammatory responses, complement activation, accelerated cell proliferation and subsequent tissue repair, and immunity directed against parasite fecundity all contributed to the development of host resistance to parasitic infection in the resistant breed

Reversible Suppression of Cyclooxygenase 2 (COX-2) Expression In Vivo by Inducible RNA Interference

Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3'untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2

The ecology of methane in streams and rivers: patterns, controls, and global significance

Streams and rivers can substantially modify organic carbon (OC) inputs from terrestrial landscapes, and much of this processing is the result of microbial respiration. While carbon dioxide (CO₂) is the major end‐product of ecosystem respiration, methane (CH₄) is also present in many fluvial environments even though methanogenesis typically requires anoxic conditions that may be scarce in these systems. Given recent recognition of the pervasiveness of this greenhouse gas in streams and rivers, we synthesized existing research and data to identify patterns and drivers of CH₄, knowledge gaps, and research opportunities. This included examining the history of lotic CH4 research, creating a database of concentrations and fluxes (MethDB) to generate a global‐scale estimate of fluvial CH₄ efflux, and developing a conceptual framework and using this framework to consider how human activities may modify fluvial CH₄ dynamics. Current understanding of CH₄ in streams and rivers has been strongly influenced by goals of understanding OC processing and quantifying the contribution of CH₄ to ecosystem C fluxes. Less effort has been directed towards investigating processes that dictate in situ CH₄ production and loss. CH₄ makes a meager contribution to watershed or landscape C budgets, but streams and rivers are often significant CH₄ sources to the atmosphere across these same spatial extents. Most fluvial systems are supersaturated with CH₄ and we estimate an annual global emission of 26.8 Tg CH₄, equivalent to ~15‐40% of wetland and lake effluxes, respectively. Less clear is the role of CH₄ oxidation, methanogenesis, and total anaerobic respiration to whole ecosystem production and respiration. Controls on CH₄ generation and persistence can be viewed in terms of proximate controls that influence methanogenesis (organic matter, temperature, alternative electron acceptors, nutrients) and distal geomorphic and hydrologic drivers. Multiple controls combined with its extreme redox status and low solubility result in high spatial and temporal variance of CH₄ in fluvial environments, which presents a substantial challenge for understanding its larger‐scale dynamics. Further understanding of CH₄ production and consumption, anaerobic metabolism, and ecosystem energetics in streams and rivers can be achieved through more directed studies and comparison with knowledge from terrestrial, wetland, and aquatic disciplines."Support for this paper was provided by funding from the North Temperate Lakes LTER program, NSF DEB‐0822700."https://esajournals.onlinelibrary.wiley.com/doi/full/10.1890/15-102

Antibodies and IL-3 support helminth-induced basophil expansion

Basophils are powerful mediators of Th2 immunity and are present in increased numbers during allergic inflammation and helminth infection. Despite their ability to potentiate Th2 immunity the mechanisms regulating basophil development remain largely unknown. We have found a unique role for isotype-switched antibodies in promoting helminth-induced basophil production following infection of mice with Heligmosomoides polygyrus bakeri or Nippostrongylus brasiliensis. H. polygyrus bakeri-induced basophil expansion was found to occur within the bone marrow, and to a lesser extent the spleen, and was IL-3 dependent. IL-3 was largely produced by CD4+CD49b+NK1.1− effector T cells at these sites, and required the IL-4Rα chain. However, antibody-deficient mice exhibited defective basophil mobilization despite intact T-cell IL-3 production, and supplementation of mice with immune serum could promote basophilia independently of required IL-4Rα signaling. Helminth-induced eosinophilia was not affected by the deficiency in isotype-switched antibodies, suggesting a direct effect on basophils rather than through priming of Th2 responses. Although normal type 2 immunity occurred in the basopenic mice following primary infection with H. polygyrus bakeri, parasite rejection following challenge infection was impaired. These data reveal a role for isotype-switched antibodies in promoting basophil expansion and effector function following helminth infection